Various conditions and diseases which manifest themselves in bone loss or thinning are a critical and growing health concern. It has been estimated that as many as 30 million Americans and 100 million worldwide are at risk for osteoporosis alone (Mundy et al. (1999) Science 286:1946-1949). Other conditions known to involve bone loss include juvenile osteoporosis, osteoporotic fractures, giant cell tumors of bone, renal osteodystrophy, osteogenesis imperfecta, hypercalcemia, hyperparathyroidism, osteomalacia, osteohalisteresis, osteolytic bone disease, osteonecrosis, Paget's disease of bone, bone loss due to rheumatoid arthritis, inflammatory arthritis, osteomyelitis, corticosteroid treatment, metastatic bone diseases or malignancy-induced osteoporosis and bone lysis, childhood idiopathic bone loss, bone fractures, periodontal bone loss, age-related loss of bone mass, osteotomy and bone loss associated with prosthetic ingrowth and other forms of osteopenia. Additionally, new bone formation is needed in many situations, e.g., to facilitate bone repair or replacement for bone fractures, bone defects, plastic surgery, dental and other implantations and in other such contexts.
Bone is a dense, specialized form of connective tissue. Bone matrix is formed by osteoblast cells located at or near the surface of existing bone matrix. Bone is resorbed (eroded) by another macrophage-type cell type known as the osteoclast. Osteoclasts secrete acids, which dissolve bone minerals, and enzymes, particularly hydrolases, which digest its organic components. Osteoclasts originate from hematopoietic precursors of the monocyte/macrophage lineage and migrate to the bone environment. Here, in the presence of the cytokines RANKL (receptor activator of NF-κB ligand) and M-CSF (macrophage colony stimulating factor), they fuse to form multinuclear cells and assume the unique osteoclast phenotype and acquire the capacity to degrade mineralized matrix [Boyle W J, Simonet W S, Lacey D L. (2003), Osteoclasts differentiation and activation. Nature 15:337-342; Teitelbaum S L. (2000), Bone resorption by osteoclasts. Science 289:1504-1508]. Osteoclasts, which are the sole bone resorbing cells, are essential for skeletal development and remodeling throughout the life of animal and man. A deficiency of osteoclasts leads to osteopetrosis, a disease manifested by increased non-remodeled bone mass, which ultimately leads to bone deformities and functional failure of other body systems. On the other hand, an increase in the number and activity of osteoclasts causes accelerated bone resorption and may lead to osteoporosis and osteolytic diseases. Thus, proper bone formation and remodeling is a dynamic process that involves an ongoing interplay between the creation and erosion activities of osteoblasts and osteoclasts (Alberts, et al., Molecular Biology of the Cell, Garland Publishing, N.Y. (3rd ed. 1994), pp. 1182-1186).
Most bone diseases are due to increased bone resorption, rendering its inhibition a primary therapeutic objective. Indeed, most osteoporosis therapies that are currently available belong in this category. Inhibition of bone resorption can be accomplished by reducing osteoclast formation and activity. These processes point to rate-limiting steps in osteoclast formation and function, and offer a number of targets for therapeutic intervention.
It has recently become clear that such molecules as receptor activator of NF-κB (RANK), RANK ligand (RANKL), and osteoprotegerin, a soluble protein that binds to RANKL and prevents its ligation to its receptor are involved in the regulation of osteoclastogenesis. RANKL stimulates osteoclastogenesis through binding to its receptor RANK on osteoclast precursors. The isolation, cloning and production of RANKL further permitted the study of osteoclastogenesis under in vitro conditions. More particularly, RANK ligand (RANKL, also known as osteoprotegerin ligand (OPGL), TNF-related activation induced cytokine (TRANCE), and osteoclast differentiation factor (ODF)), expressed on stromal cells, osteoblasts, activated T-lymphocytes and mammary epithelium, is the unique molecule essential for differentiation of macrophages into osteoclasts (Lacey, et al. (1998) Cell 93: 165-176). The cell surface receptor for RANKL is RANK, Receptor Activator of Necrosis Factor (NF)-kappa B. RANKL is a type-2 transmembrane protein with an intracellular domain of less than about 50 amino acids, a transmembrane domain of about 21 amino acids, and an extracellular domain of about 240 to 250 amino acids. RANKL exists naturally in transmembrane and soluble forms. The deduced amino acid sequence for at least the murine, rat and human forms of RANKL and variants thereof are known. (See e.g., Anderson, et al., U.S. Pat. No. 6,017,729, Boyle, U.S. Pat. No. 5,843,678, and Xu J. et al., J. Bone Min. Res. (2000) 15:2178) which are incorporated herein by reference in their entirety. RANKL (OPGL) has been identified as a potent inducer of bone resorption and as a positive regulator of osteoclast development. (Lacey et al., supra.).
RANK signaling, activated by its ligand RANKL, which is expressed on stromal cells and osteoblasts [Suda T, Takahashi N, Udagawa N, Jimi E, Gillespie M T, Martin T J. (1999), Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr Rev 20:345-357], is mediated by a series of protein kinases including c-Src, c-Jun N terminal kinase (JNK), p 38, extracellular signal related kinase (ERK), phosphoinositol-3-kinase (PI-3K), and those activating NF-κB [Darnay B G, Haridas V, Ni J, Moore P A, Aggarwal B B, (1998), Characterization of the intracellular domain of receptor activator of NF-kappaB (RANK). Interaction with tumor necrosis factor receptor-associated factors and activation of NF-kappaβand c-Jun N-terminal kinase. J Biol Chem 273:20551-2055; Galibert L, Tometsko M E, Anderson D M, Cosman D, Dougall, W C. (1998), The involvement of multiple tumor necrosis factor receptor (TNFR)-associated factors in the signaling mechanisms of receptor activator of NF-kappaB, a member of the TNFR superfamily. J Biol Chem 273:34120-34127; Lee S E, Woo K M, Kim S Y, Kim H-M, Kwack K, Lee Z H, Kim H-H. (2002), The phosphatidylinositol 3-Kinase, p 38, and extracellular signal-regulated kinase pathways are involved in osteoclast differentiation. Bone 30:71-77; Matsumoto M, Sudo T, Saito T, Osada A, Tsujimoto M. (2000), Involvement of p38 mitogen-activated protein kinase signaling pathway in osteoclastogenesis mediated by receptor activator of NF-kappa B ligand (RANKL). J Biol Chem 275:31155-31161]. M-CSF, which via, its receptor, c-Fms, stimulates many of the same pathways, promotes proliferation of osteoclast precursors and survival of the mature resorptive cell [Tanaka S, Takahashi N, Udagawa N, Tamura T, Akatsu T, Stanley E R, Kurokawa T, Suda T. (1993), Macrophage colony-stimulating factor is indispensable for both proliferation and differentiation of osteoclast progenitors. J Clin Invest 91:257-263; Woo K M, Kim H M, Ko J S. (2002), Macrophage colony-stimulating factor promotes the survival of osteoclast precursors by up-regulating Bcl-XL. Exp Mol Med 34:340-346]. Together, therefore, RANKL and M-CSF induce expression of genes, such as those encoding tartrate-resistant acid phosphatase (TRAP), cathepsin K (CATK), calcitonin receptor and β3 integrin, which characterize the mature osteoclast and its committed precursors [Faccio R, Zallone A, Ross F P, Teitelbaum S L. (2003), c-Fms and the avb3 integrin collaborate during osteoclast differentiation. J Clin Invest 111:749-758; Kudo O, Sabokbar A, Pocock A, Itonaga I, Athanasou N A. (2002). Isolation of human osteoclasts formed in vitro: hormonal effects on the bone-resorbing activity of human osteoclasts. Calcif Tissue Int 71:539-546]
The fact that osteoclasts are derived from macrophages, which are professional phagocytes of myeloid origin that reside in all tissues and organs and are cells that are fundamental to immune recognition, has led to a series of experiments which link the immune system to osteoclast recruitment and function. For example, T-lymphocycte-produced cytokines, including RANKL and TNFα, appear central to the enhanced osteoclastogenesis responsible for the bone loss attending menopause and the peri-articular bone erosions of rheumatoid arthritis [Cenci S, Weitzmann M N, Roggia C, Namba N, Novack D, Pacifici R. (2000), Estrogen deficiency induces bone loss by enhancing T cell production of TNFα. J Clin Invest 106:1229-1237; Romas E, Gillespie M T, Martin T J. (2002), Involvement of receptor activator of NFκB ligand and tumor necrosis factor-alpha in bone destruction in rheumatoid arthritis. Bone 30:340-346; Weitzmann M N, Cenci S, Rifas L, Brown. C., Pacifici R. (2000), IL-7 stimulates osteoclast formation by upregulating the T-cell production of soluble osteoclastogenic cytokines. Blood 96:1873-1878]. In this context, the process of antigen presentation, itself, is also a fundamental event in pathological osteoclastogenesis [Jenkins J K, Hardy K J, McMurray RW. (2002), The pathogenesis of rheumatoid arthritis: a guide to therapy. Am J Med Sci 323:171-180]. Under appropriate conditions, macrophages may fuse with each other to form multinucleate giant cells which are descriptively separated into foreign body and Langerhans giant cells by the organization of the nuclei within the cell. In bone, macrophages fuse to form osteoclasts which mediate bone resorption (Johansson, B., Halldner, L., Dunwiddie, T. V., Masino, S. A., Poelchen, W., Gimenez-Llort, L., Escorihuela, R. M., Femandez-Teruel, A., Wiesenfeld-Hallin, Z., Xu, X. J., et al. (2001), Hyperalgesia, anxiety, and decreased hypoxic neuroprotection in mice lacking the adenosine A1 receptor. Proc Natl Acad Sci USA 98:9407-9412) both in normal day-to-day bone remodeling and in such pathologic situations as osteoporosis, inflammatory arthritis with bony erosions, peri-prosthetic bone resorption, hypercalcemia of malignancy and bone metastases (Gimenez-Llort, L., Fernandez-Teruel, A., Escorihuela, R. M., Fredholm, B. B., Tobena, A., Pekny, M., and Johansson, B. (2002), Mice lacking the adenosine A1 receptor are anxious and aggressive, but are normal learners with reduced muscle strength and survival rate. Eur J Neurosci 16:547-550). Fusion of macrophages is critical for the differentiation of osteoclasts, as mononuclear macrophages cannot resorb bone efficiently.
The major characteristics of osteoclasts which differentiate them from other forms of giant cells include: tartrate-resistant acid phosphatase (TRAP) staining (shared with macrophages), multinuclearity, formation of actin ring structure and a polar cell body during resorption, and contraction in response to calcitonin, expression of molecular markers including the calcitonin receptor, RANK (receptor of RANKL, receptor activator of NFκB ligand), c-fms (receptor of M-CSF, macrophage-colony stimulating factor), cathepsin K, c-src, fosL1 and the vitronectin receptor (integrin αvβ3).
Adenosine is a nucleoside that occurs naturally in mammals, which acts as a ubiquitous biochemical messenger. The heart, for instance, produces and releases adenosine in order to modulate heart rate and coronary vasodilation. Likewise, adenosine is produced in the kidney to modulate essential physiological responses, including glomerular filtration rate (GFR), electrolyte reabsorption, and renin secretion.
Adenosine is known to bind to and activate seven-transmembrane spanning G-protein coupled receptors, thereby eliciting a variety of physiological responses. There are 4 known subtypes of adenosine receptors (i.e., A1, A2a, A2b, and A3), which mediate different, and sometimes opposing, effects. For example, activation of the adenosine A1 receptor, elicits an increase in renal vascular resistance, which leads to a decrease in glomerular filtration rate (GFR), while activation of the adenosine A2a receptor elicits a decrease in renal vascular resistance. Conversely, blockade of the A1 adenosine receptor decreases afferent arteriole pressure, leading to an increase in GFR and urine flow, and sodium excretion. Furthermore, A2A adenosine receptors modulate coronary vasodilation, whereas A2B receptors have been implicated in mast cell activation, asthma, vasodilation, regulation of cell growth, intestinal function, and modulation of neurosecretion (See Adenosine A2B Receptors as Therapeutic Targets, Drug Dev Res 45:198; Feoktistov et al., Trends Pharmacol Sci 19:148-153 and Ralevic, V and Burnstock, G. (1998), Pharmacological Reviews, Vol. 50: 413-492), and A3 adenosine receptors modulate cell proliferation processes. Two receptor subtypes (A1 and A2a) exhibit affinity for adenosine in the nanomolar range while two other known subtypes A2b and A3 are low-affinity receptors, with affinity for adenosine in the low-micromolar range. A1 and A3 adenosine receptor activation can lead to an inhibition of adenylate cyclase activity, while A2a and A2b activation causes a stimulation of adenylate cyclase.
Diseases that can be prevented and/or treated with A1 adenosine receptor antagonists include diseases and disorders wherein activation of A1 adenosine receptors plays a role in pathophysiology. For example, A1 adenosine receptor antagonists are useful for treating cognitive disorders and dementias such as Alzheimers disease, and for treating stress, depression, cardiac arrhythmia, restoration of cardiac function, congestive heart failure, asthma, and respiratory disorders (e.g., bronchial asthma, allergic lung diseases). They also reduce ischemia-induced injury of the brain, heart and kidney. A1 adenosine receptor antagonists have pronounced effects on the kidney, and have shown to be potent diuretics and natriuretics with little effective on potassium excretion. Thus, they are also renal protective, useful for the treatment of renal failure, renal dysfunction, nephritis, hypertension, and edema. It has been suggested that A2a antagonists may be beneficial for patients suffering from Morbus Parkinson (Parkinson's disease). Adenosine receptor antagonists may also be valuable for treatment of allergic inflammation and asthma. Available information (for example, Nyce & Metzger “DNA antisense Therapy for Asthma in an Animal Model” Nature (1997) 385: 721-5) indicates that in this pathophysiologic context, A1 antagonists may block contraction of smooth muscle underlying respiratory epithelia, while A2b or A3 receptor antagonists may block mast cell degranulation, mitigating the release of histamine and other inflammatory mediators. A2b receptors have been discovered throughout the gastrointestinal tract, especially in the colon and the intestinal epithelia. It has been suggested that A2b receptors mediate cAMP response (Strohmeier et al., J. Bio. Chem. (1995) 270:2387-94).
There is a need for new agents that are effective for treating subjects suffering from conditions characterized by loss of bone, or for treating subjects at risk for developing such conditions. There is also a need for more effective treatment strategies for increasing bone mass in subjects suffering from diseases, disorders, or conditions that lead to decreased bone density. These needs are addressed by the agents and methods of the present invention.
All publications, patent applications, patents and other reference material mentioned are incorporated by reference in their entirety. In addition, the materials, methods and examples are only illustrative and are not intended to be limiting. The citation of references herein shall not be construed as an admission that such is prior art to the present invention.